The invention relates generally to non-naturally occurring novel proteins which have insecticidal properties, and more specifically to the design, preparation, and use of proteins that have been deallergenized while maintaining their insecticidal properties. Deallergenized patatin proteins include variants that have had allergenic sequences modified, and permuteins that have had their amino acid sequences rearranged at one or more breakpoints.
Insecticidal Proteins
The use of natural products, including proteins, is a well known method of controlling many insect, fungal, viral, bacterial, and nematode pathogens. For example, endotoxins of Bacillus thuringiensis (B.t.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these endotoxins have been introduced into and expressed by various plants, including cotton, tobacco, and tomato. There are, however, several economically important insect pests such as boll weevil (BWV), Anthonomus grandis, and corn rootworm (CRW), Diabrotica spp. that are not as susceptible to B.t. endotoxins as are insects such as lepidopterans. In addition, having other, different gene products for control of insects which are susceptible to B.t. endotoxins is important, if not vital, for resistance management.
It has been recently discovered that the major storage protein of potato tubers, patatins (Gaillaird, T., Biochem. J. 121: 379-390, 1971; Racusen, D., Can. J. Bot., 62: 1640-1644, 1984; Andrews, D. L., et al., Biochem. J., 252: 199-206, 1988), will control various insects, including western rootworm (WCRW, Diabrotica virigifera), southern corn rootworm (SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomus grandis) (U.S. Pat. No. 5,743,477). Patatins are lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed, resulting in no reproduction. These proteins, have nonspecific lipid acyl hydrolase activity and studies have shown that the enzyme activity is essential for its insecticidal activity (Strickland, J. A., et al., Plant Physiol., 109: 667-674, 1995; U.S. Pat. No. 5,743,477). Patatins can be applied directly to the plants or introduced in other ways well known in the art, such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
In potato, the patatins are found predominantly in tubers, but also at much lower levels in other plant organs (Hofgen, R. and Willmitzer, L., Plant Science, 66: 221-230, 1990). Genes that encode patatins have been previously isolated by Mignery, G. A., et al. (Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, G. A., et al., Gene, 62: 27-44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255-269, 1988) and others. Patatins are found in other plants, particularly solanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501-509, 1991; Vancanneyt, et al., Plant Cell, 1: 533-540, 1989) and recently Zea mays (WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155-1159, 1987) transferred it to tobacco plants, and observed expression of patatin, demonstrating that the patatin genes can be heterologously expressed by plants.
Patatin is an attractive for use in planta as an insect control agent, but unfortunately a small segment of the population displays allergic reactions to patatin proteins, and in particular to potato patatin, as described below.
Food Allergens
There are a variety of proteins that cause allergic reactions. Proteins that have been identified as causing an allergic reaction in hypersensitive patients occur in many plant and animal derived foods, pollens, fungal spores, insect venoms, insect feces, and animal dander and urine (King, H. C., Ear Nose Throat J., 73(4): 237-241, 1994; Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995; Astwood, J. D. and Fuchs R. L., Monographs in allergy Vol. 32: Highlights in food allergy, pp. 105-120, 1996; Metcalfe, D. D., et al., Critical Reviews in Food Science and Nutrition, 36S: 165-186, 1996 ). The offending proteins of many major sources of allergens have been characterized by clinical and molecular methods. The functions of allergenic proteins in vivo are diverse, ranging from enzymes to regulators of the cell cytoskeleton.
To understand the molecular basis of allergic disease, the important IgE binding epitopes of many allergen proteins have been mapped (Elsayed, S. and Apold, J., Allergy 38(7): 449-459, 1983; Elsayed, S. et al., Scand J. Clin. Lab. Invest. Suppl. 204: 17-31 1991; Zhang, L., et al., Mol. Immunol. 29(11): 1383-1389, 1992). The optimal peptide length for IgE binding has been reported to be between 8 and 12 amino acids. Conservation of epitope sequences is observed in homologous allergens of disparate species (Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995). Indeed, conservative substitutions introduced by site-directed mutagenesis reduce IgE binding of known epitopes when presented as peptides.
Food allergy occurs in 2-6% of the population. Eight foods or food groups (milk, eggs, fish, crustacea, wheat, peanuts, soybeans, and tree nuts) account for 90% of allergies to foods. Nevertheless, over 160 different foods have been reported to cause adverse reactions, including potato (Hefle, S., et al., Crit. Rev. in Food Sci. Nutr., 36S: 69-90, 1996).
Mode of Action of Allergens
Regardless of the identity of the allergen, it is theorized that the underlying mechanism of allergen response is the same. Immediate hypersensitivity (or anaphylactic response) is a form of allergic reaction which develops very quickly, i.e., within seconds or minutes of exposure of the patient to the causative allergen, and is mediated by B lymphocyte IgE antibody production. Allergic patients exhibit elevated levels of IgE, mediating hypersensitivity by priming mast cells which are abundant in the skin, lymphoid organs, in the membranes of the eye, nose and mouth, and in the respiratory tree and intestines. The IgE in allergy-suffering patients becomes bound to the IgE receptors of mast cells. When this bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and release various substances such as histamine into the surrounding tissue (Church et al. In: Kay, A. B. ed., Allergy and Allergic Diseases, Oxford, Blackwell Science, pp. 149-197, 1997).
It is the release of these substances which is responsible for the clinical symptoms typical of immediate hypersensitivity, namely contraction of smooth muscle in the airways or in the intestine, the dilation of small blood vessels, and the increase in their permeability to water and plasma proteins, the secretion of thick sticky mucus, and (in the skin) the stimulation of nerve endings that result in itching or pain. Immediate hypersensitivity is, at best, a nuisance to the suffer; at worst it can present very serious problems and can in rare cases even result in death.
Allergic Reactions to Potato
Food allergy to potato is considered rare in the general population (Castells, M. C., et al., Allergy Clin. Immunol., 8: 1110-1114, 1986; Hannuksela, M., et al., Contact Dermatitis, 3: 79-84, 1977; Golbert, T. M., et al., Journal of Allergy, 44: 96-107, 1969). Approximately 200 individuals have participated in published clinical accounts of potato allergy (Hefle, S. et al., Critical Reviews in Food Science and Nutrition, 36S: 69-90, 1996). A number of IgE binding proteins have been identified in potato tuber extracts (see Table 1), however the amino acid sequence and function of these proteins has not been determined (Wahl, R., et al., Intl. Arch. Allergy Appl. Immunol., 92: 168-174, 1990).
Improved Safety From the use of Hyloallergenic Proteins
Patatin has been identified as an allergenic protein (Seppala, U. et al., J. Allergy Clin. Immunol. 103:165-171, 1999). Accordingly, potato allergic subjects may not be able to safely consume products containing unmodified patatin protein, such as crops to which foliar applications of patatins have been applied, or crops which have been engineered to express patatin. In addition, proliferation of food allergens in the food supply is considered hazardous (Metcalfe, D. D., et al., Critical Reviews and Food Science and Nutrition, 36S: 165-186, 1996). There are additional concerns regarding the use of potentially allergenic food proteins where workers might be exposed to airborne particulates, initiating a new allergic response (Moneret-Vautrin, D. A., et al., Rev. Med. Interne., 17(7): 551-557, 1996).
Permuteins
Novel proteins generated by the method of sequence transposition resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J. Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem., 204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991; MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitro application of sequence rearrangement to proteins was described by Goldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion or sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids and represent a broad range of structural classes (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., Nature Struct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31: 1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, et al., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad. Sci U.S.A., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243: 206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al., Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4: 994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551, 1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al., Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman, et al., Proc. Natl. Acad Sci., 91: 6889-6893, 1994).
There exists a need for the development of plant expressible insecticidal proteins which possess minimal or no allergenic properties.
Novel protein sequences, and nucleic acid sequences encoding them are disclosed. The proteins maintain desirable enzymatic and insecticidal properties while displaying reduced or eliminated allergenicity.
Allergenic epitopes are identified by scanning overlapping peptide sequences with an immunoreactivity assay. Alanine scanning and xe2x80x98rational substitutionxe2x80x99 is performed on identified peptide sequences to determine specific amino acids which contribute to antibody binding, and presumably, to the allergenic properties of the whole protein. Individual mutations are introduced into the whole protein sequence by methods such as site directed mutagenesis of the encoding nucleic acid sequence to delete or modify the allergenic sequences.
Glycosylation target residues are identified within amino acid sequences of proteins which have demonstrated allergy eliciting properties. Glycosylation target amino acid residues are rationally substituted with other amino acid residues to eliminate glycosylation and to provide a variant deglycosylated protein. The variant protein may then exhibit reduced allergen eliciting properties and may also exhibit reduced binding to IgE within serum of patients observed to be allergic to said glycosylated protein.
Permuteins of the deallergenized protein sequences can be constructed to further reduce or eliminate allergic reactions. The encoding nucleic acid sequence is modified to produce a non-naturally occurring protein having a linear amino acid sequence different from the naturally occurring protein sequence, while maintaining enzymatic and insecticidal properties. The permutein is preferably produced in plant cells, and more preferably produced at a concentration which is toxic to insects ingesting the plant cells.
Methods for reducing, eliminating, or decreasing allergen eliciting properties of a protein are specifically contemplated herein. Such methods comprise steps including identifying one or more patients exhibiting an allergic sensitivity to an allergen eliciting protein and obtaining a sample of serum from the patient; exposing the patient serum to a first set of synthetic overlapping peptides which represent the allergen eliciting protein in order to identify such peptides which exhibit epitopes which bind to IgE present within the allergic patients"" serum and wherein the IgE present in the serum has a specific affinity for the said allergen eliciting protein; producing a second set of peptides which are variant peptides based on the first set of peptides which were identified to bind specifically to IgE present in patient serum, wherein the second set variant peptides exhibit alanine scanning or rational scanning amino acid substitutions which exhibit reduced, decreased, or eliminated IgE binding when compared to the first set non-variant peptides, and wherein such substitutions which reduce, eliminate or decrease IgE binding are identified as result effective substitutions; and modifying the amino acid sequence of the allergen eliciting protein to contain one or more of said result effective substitutions, wherein the modified protein is a variant of the allergen eliciting protein which lacks allergen eliciting protein or exhibits reduced allergen eliciting properties, and wherein the variant of the allergen eliciting protein comprising one or more result effective substitutions exhibits reduced, decreased, or totally eliminated binding of IgE present within said patients"" serum.
The novel proteins can be used in controlling insects, as nutritional supplements, in immunotherapy protocols, and in other potential applications. Transgenic plant cells and plants containing the encoding nucleic acid sequence can be particularly beneficial in the control of insects, and as a nutritional/immunotherapy material.